ORIGINAL ARTICLE

DNA sequencing of maternal plasma to identify Down syndromeand other trisomies in multiple gestations†

Jacob A. Canick1*, Edward M. Kloza1, Geralyn M. Lambert-Messerlian1, James E. Haddow1, Mathias Ehrich2, Dirk van den Boom2,Allan T. Bombard2,3,4, Cosmin Deciu3 and Glenn E. Palomaki1

1Division of Medical Screening and Special Testing, Department of Pathology and Laboratory Medicine, Women and Infants Hospital, Alpert Medical School ofBrown University, Providence, RI, USA2Sequenom, Inc., San Diego, CA, USA3Sequenom Center for Molecular Medicine, San Diego, CA, USA4Department of Reproductive Medicine, University of California San Diego, San Diego, CA, USA*Correspondence to: Jacob A. Canick. E-mail: jcanick@wihri.org† This article was published online on May 14, 2012. Errors were subsequently identified in the Abstract. This notice is included in the online and print versions toindicate that both have been corrected [June 6, 2012].

ABSTRACTObjective Studies on prenatal testing for Down syndrome (trisomy 21), trisomy 18, and trisomy 13 by massively parallelshotgun sequencing (MPSS) of circulating cell free DNA have been, for the most part, limited to singleton pregnancies. IfMPSS testing is offered clinically, it is important to know if these trisomies will also be identified in multiple pregnancies.

Method Among a cohort of 4664 high-risk pregnancies, maternal plasma samples were tested from 25 twin pregnancies(17 euploid, five discordant and two concordant for Down syndrome; one discordant for trisomy 13) and two euploid tripletpregnancies [Correctionmade here after initial online publication.]. Results were corrected for GC content bias. For eachtarget chromosome (21, 18, and 13), z-scores of 3 or higher were considered consistent with trisomy.

Results Seven twin pregnancies with Down syndrome, one with trisomy 13, and all 17 twin euploid pregnancies werecorrectly classified [detection rate 100%, 95% confidence interval (CI) 59%–100%, false positive rate 0%, 95%CI 0%–19.5%],as were the two triplet euploid pregnancies.

Conclusion Although study size is limited, the underlying biology combined with the present data provide evidencethat MPSS testing can be reliably used as a secondary screening test for Down syndrome in women with high-risk twingestations. © 2012 John Wiley & Sons, Ltd.

Funding sources: Sequenom, Inc. fully funded the project through a grant to Women & Infants Hospital of Rhode Island. Sequenom Center for Molecular Medicine(SCMM) was responsible for developing an internally validated laboratory developed test (LDT) for detecting Down syndrome in maternal plasma using massivelyparallel shotgun sequencing and for providing clinical interpretation of the test results. SCMM also identified, equipped and trained an independent laboratory totest a subset of samples through a separate contract with UCLA. The sponsor did not control study design, identify or communicate with Enrollment Sites, thaw or testsamples prior to the formal testing period, have access to patient information prior to all testing being completed, analyze study results, prepare drafts of the manuscript, orhave final decisions on manuscript content.Conflicts of interest: Palomaki and Canick were members of the Sequenom Clinical Advisory Board for 6 months, and resigned when the study was funded in 2008.Van den Boom, Ehrich, and Bombard are employees and shareholders of Sequenom, Inc. Deciu is an employee of Sequenom Center for Molecular Medicine anda shareholder of Sequenom, Inc.

INTRODUCTIONThe identification of Down syndrome pregnancy by massivelyparallel shotgun sequencing (MPSS) of circulating cell freeDNA in maternal plasma is highly effective. To date, studiesusing MPSS have provided results in more than 300 singletonpregnancies in which Down syndrome was caused by an extracopy of the number 21 chromosome in all cells examined(so-called ‘classical’ Down syndrome; karyotype: 47,XX,+21 or 47,XY,+21),1–5 and in one case, due to an unbalanced Robertsoniantranslocation (46,XY,der(14;21)(q10;q10),+21).6 Recent studies

have also shown that singleton pregnancies affected by trisomies18 and 13 can be reliably identified by MPSS.1,7,8

Twin pregnancies are considerably less common thansingleton, with a live birth rate in the United States in 2009 of1 in 30 deliveries (http://www.cdc.gov/nchs/fastats/multiple.htm). Multiple gestations are becoming more prevalent due tothe increased use of assisted reproductive technologies. Amongaffected monozygous twins, discordance for a trisomy is rare,but among affected dizygous twins, the fetuses are nearly alwaysdiscordant. Given that identification of Down syndrome by

Prenatal Diagnosis 2012, 32, 730–734 © 2012 John Wiley & Sons, Ltd.

DOI: 10.1002/pd.3892

MPSS of maternal plasma relies on a small incremental increasein the proportion of DNA fragmentsmapped to chromosome 21,it is uncertain whether Down syndrome (or other trisomies) intwins or other multiples will be reliably detected. A previouslypublished study, by Sehnert et al.8, included five twinpregnancies, four in a training set, and one in a test set for anew massively parallel testing method. One was presumablymonozygotic and one dizygotic for Down syndrome, and threeothers were presumably euploid; all five were apparentlycorrectly categorized by massively parallel sequencing.

As part of an international collaborative validation study forMPSS testing in which maternal plasma samples werecollected prior to diagnostic testing from over 4000 womenwith pregnancies at high risk for trisomy, multiple gestationswere included. The current study explores the results oftesting in those multiple pregnancies.

MATERIALS AND METHODSWomen with a pregnancy at high risk for Down syndromebased on age, family history, or a positive serum and/orsonographic screening test provided consent, plasma samples,and demographic and pregnancy-related information underlocal institutional review board approval as part of amulticenter study (www.clinicaltrials.gov NCT00877292).Information regarding the use of assisted reproductivetechnologies was not collected. Samples were drawnimmediately prior to invasive testing (chorionic villus samplingor amniocentesis), processed within 6h, stored at �80 �C andshipped on dry ice to the Coordinating Center at Women andInfants Hospital, Rhode Island, where samples were keptfrozen until tested at a Clinical Laboratory ImprovementAmendments-certified laboratory (Sequenom Center forMolecular Medicine, San Diego, CA); now also accredited bythe College of American Pathologists. Karyotype data on eachpregnancy were obtained from the enrollment sites and keptat the coordinating center site in Maine. In 0.3% of allpregnancies, only quantitative fluorescent–polymerase chainreaction results were available due to diagnostic standards inplace at four sites. Enrollment sites, site investigators, oversightcommittee, and other study personnel have been cited in theprimary publication.5 Samples used in this study were testedwithout knowledge of the karyotype, gestational age, orgeographic origin of the sample. A subset of results for singletonpregnancies was validated at an independent academiclaboratory at theUniversity of California, Los Angeles.5,7 Samplesobtained from all multiple gestations with Down syndrome,trisomy 18, or trisomy 13 were submitted for analysis, along withall euploid triplet pregnancies and a random selection of euploidtwin pregnancies (22% of a total of 77).

Testing was by MPSS, during a single 9 week time period asdescribed previously.3,5 In brief, circulating cell-free DNAfragments were isolated from 4mL of maternal plasma andquantified with an assay that determines the fetal contribution(fetal fraction).1 The remaining isolate was used to generatesequencing libraries, which were normalized and multiplexedto allow four samples to be run in a single flow cell lane (eightlanes per flow cell, allowing for up to 32 patient samples perflow cell). The DNA libraries were prepared and quality

controlled using a microfluidics platform (Caliper Life Sciences,Hopkinton, MA) and clusters generated using the cBot platform(Illumina, Inc, San Diego, CA). Flow cells were sequenced onthe Illumina HiSeq 2000; data were analyzed using Illuminasoftware, and a robust estimate of the standard deviations aboveor below the central estimate (z-score) was calculated forchromosomes 21, 18, and 13. Z-scores at or above 3 werereported to be consistent with the presence of trisomy. All threechromosome results were corrected for the guanine�cytosinecontent bias, and the chromosome 21 results were also repeat-masked, post alignment. The MPSS results from the multiplegestations were compared with the reference range establishedfor the interpretation of singleton pregnancies. The SequenomCenter for Molecular Medicine did not know whether thesamples were from singleton or multiple gestations.

RESULTSAmong the 4644 pregnancies enrolled, seven twin pregnanciesaffected by Down syndrome were identified by theirkaryotypes; two in which both fetuses were affected and fivein which just one fetus was affected. One of the concordantDown syndrome cases was due to an unbalanced 14;21translocation. One twin pregnancy discordant for trisomy 13was also identified. MPSS testing was performed on maternalplasma samples from all eight cases of trisomy, along withcontrol samples from 17 euploid twin pregnancies (randomlyselected from the 62 eligible twin pregnancies) and the onlytwo eligible euploid triplet pregnancies (Table 1).

Figure 1 shows the chromosome 21, 18, and 13 results,expressed as robust z-scores versus the fetal fraction for the27 multiple gestations. The upper limit of euploid is set at az-score of +3. As a reference, the median chromosome 21 z-score (after alignment with a repeat-masked genome andaccounting for guanine�cytosine content bias) for the euploidsingleton pregnancies reported previously5,7 was 0.013 with99.8% of the samples below a z-score of 3. For chromosomes18 and 13, the median z-scores and proportion of samplesbelow a z-score of 3 for the euploid singleton pregnancieswas 0.000, 99.0% and 0.000, 99.7%, respectively. For all samplegroups, approximately half of the gestational ages were fromthe late first trimester (9–14 completed weeks) and half fromthe early second trimester (15–21 completed weeks).

Chromosome 21 z-scores were elevated in all seven twinpregnancies affected by Down syndrome (100% detection rate,95% CI 59%–100%), with a median of 12.3 (range: 5.3–17.4)(Table 1). Among the five twin pregnancies discordant forDown syndrome, chromosome 21 z-scores (range 5.3–17.4)were similar to those from the two concordant twinpregnancies (12.3, 13.2). Chromosome 18 and 13 z-scores wereunder 3 for all seven of the twin pregnancies affected byDown syndrome. The twin pregnancy discordant for trisomy13 had a chromosome 13 z-score of 5.1, with chromosome 21and 18 z-scores of 0.3 and 0.1, respectively.

Median z-scores for chromosomes 21, 18, and 13 in the 17euploid twin pregnancies were �0.1, 0.1, and �0.2, respectively.No z-score in euploid twins was over 3 (0% false positive rate,95% CI 0%–21%). The two euploid triplet pregnancies hadunremarkable chromosomes 21, 18, and 13 z-scores (�0.1, �0.7,

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�0.6 and �0.1, �0.6, 0.4, respectively). There was no obviousdifference in z-scores based on sex chromosome concordance ordiscordance (Table 1). For each of the sets of chromosome testresults, the case and control z-score results were plotted againstfetal fraction (Figure 1). Figure 1A (chromosome 21 results)shows the expected relationship between increasing z-score inDown syndrome pregnancies versus increasing fetal fraction.There were insufficient cases available to explore thisrelationship for trisomy 18 (none) or trisomy 13 (one) in twins.

To help understand why z-scores among discordant Downsyndrome twin pregnancies were in the same range as bothconcordant twin and classical singleton Down syndromepregnancies, we compared the fetal fraction of DNA in thematernal plasma in twin pregnancies with the fetal fraction insingleton pregnancies. The fetal fraction in 17 euploid twinpregnancies [geometric mean 18.1%, average 20.2%, logstandard deviation (SD) 0.208] was significantly higher thanthe geometric mean of 13.4% (log SD 0.205) for 1471 euploid

singleton pregnancies (t= 2.59, p= 0.0097, after logarithmictransformation). The fetal fraction in the seven Downsyndrome pregnancies was 17.8% (geometric mean), nearlythe same as for all twin pregnancies (18.1%). The fetal fractionfor the trisomy 13 twin case was 7%, the lowest fetal fraction fora twin pregnancy sample undergoing testing.

DISCUSSIONIn the present study, MPSS testing correctly identified all seventwin pregnancies with Down syndrome and the one twinpregnancy with trisomy 13, indicating that this test can beclinically useful in testing twin pregnancies. On average,multiple pregnancies in the present study contributed 35%more fetal DNA than singleton pregnancies to the total freeDNA in maternal plasma (18.1% vs 13.4%). Twin pregnancies,both monozygous and dizygous, have a higher placental massthan singleton pregnancies9 and therefore would be expectedto have a higher fetal fraction due to more fetal DNA entering

Table 1 Gestational age and DNA test results in multiple pregnancies with and without a common autosomal trisomy

Number of fetuses and their karyotypesGestationalage (weeks) Fetal fraction (%)

Chromosome z-score

Fetus 1 Fetus 2 Fetus 3 21 18 13

47,XX,+21 47,XX,+21 — 13.6 15 13.2 0.6 �2.9

46,XX,t(14;21) 46,XX,t(14;21) — 12.7 11 12.3 �1.2 �0.5

47,XX,+21 46,XX — 13.3 16 5.3 �0.9 0.6

47,XX,+21 46,XX — 18.6 12 10.1 0.7 0.6

47,XY,+21 46,XX — 15.7 41 14.5 �0.8 �1.5

47,XY,+21 46,XX — 19.0 21 17.4 �0.1 �0.5

47,XY,+21 46,XX — 12.1 21 5.3 1.0 2.99

Mean 15.0 19.6 11.2 �0.1 �0.2

47,XY,+13 46,XX — 16.6 7 0.3 0.1 5.1

46,XX 46,XX — 11.7 42 �0.1 0.9 �1.2

46,XX 46,XX — 12.0 10 0.0 0.9 �0.5

46,XX 46,XX — 13.7 29 �0.8 �1.2 �0.9

46,XX 46,XX — 12.1 18 0.6 1.8 2.3

46,XX 46,XX — 16.0 14 0.1 1.5 1.9

46,XX 46,XX — 15.0 18 �0.2 1.4 �0.6

46,XX 46,XY — 10.9 39 1.5 0.3 0.3

46,XX 46,XY — 12.9 22 �0.8 0.0 �0.1

46,XX 46,XY — 15.6 23 �0.1 �0.7 �1.4

46,XX 46,XY — 17.7 18 0.3 0.1 �0.1

46,XX 46,XY — 16.9 9 0.9 �0.2 �0.9

46,XY 46,XY — 18.0 15 �2.3 �0.6 �0.5

46,XY 46,XY — 16.1 27 0.1 0.4 0.7

46,XY 46,XY — 14.0 11 0.0 �0.7 0.0

46,XY 46,XY — 16.1 21 �0.4 �1.7 �0.7

46,XY 46,XY — 17.0 20 �0.6 �0.2 �0.5

46,XY 46,XY — 15.2 7 0.1 0.1 �1.9

Mean 14.8 20.2 �0.1 0.1 �0.2

46,XX 46,XX 46,XY 16.0 55 0.1 �0.7 �0.6

46,XX 46,XY 46,XY 16.0 18 �0.1 �0.6 0.4

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the maternal circulation. An association between increasingfetal fraction and increasing chromosomes 21, 18, and 13z-scores in singleton affected pregnancies has been clearlyshown.1,6,7

Two factors could potentially influence the reliability oftesting in twin pregnancies. First, the higher fetal fractionin twin pregnancies (euploid or Down syndrome) would beexpected to lead to better separation between Downsyndrome and euploid z-scores in twin compared withsingleton pregnancies. However, a second factor might reducethis advantage. Because most affected singleton pregnancieswill be discordant for Down syndrome and assuming thateach fetus contributes equally to the circulating cell-freeDNA, then the ratio of fetal to maternal of 1 : 9 (10%) in adiscordant twin pregnancy is effectively a ratio of trisomic todisomic material of 0.5 : 9.5 (1 : 19 or 5%). When these twofactors are taken together, the effective fetal fraction in twinsdiscordant for Down syndrome would be about 6.8%, roughlytwo-thirds that of euploid singleton pregnancies (10%). This isan especially important finding when the fetal fractionapproaches the lower limit of acceptability for singletonpregnancies (4%). The equivalent limit for discordant twinswould be at about 6%. For twin samples falling between4% and 6%, interpretations would need to rely more onthe laboratory director’s judgment. In contrast, when bothtwins are affected by Down syndrome, the observed fetalfraction and effective fetal fraction are equivalent, and theinterpretation can be made as though it were a singletonpregnancy. When MPSS test results are consistent with a fetaltrisomy in twin pregnancies, however, amniocentesis of eachfetal sac is necessary to conclusively determine concordanceversus discordance. The 35% higher fetal fraction associatedwith twin pregnancies cannot, by itself, reliably identifymultiple gestations.

In the present study, the chromosome 21 z-score was high inone twin pregnancy concordant for Down syndrome with achromosome 14;21 translocation. This reinforces thattranslocation Down syndrome will be identified through MPSStesting in twin pregnancies, as was previously shown for thesame karyotype in a singleton pregnancy.6 A recent study10

used DYS14 Y-specific marker levels to conclude that fetalfractions in 29 twins with two male fetuses were higher thanin the 36 twins with one male fetus. The majority of samplingoccurred after 20weeks’ gestation. Our numbers were muchsmaller; we found that the six twins with two male fetuseshad the lowest fetal fraction (geometric mean 15.4%)compared with the five with one male (20.0%) or the six withtwo female fetuses (19.5%).

The cohort of pregnancies used in this study was largeenough to include only a modest number of aneuploid twinpregnancies. Because Down syndrome is more commonthan either trisomy 18 or trisomy 13, there were seven twinpregnancies with one or both fetuses having Downsyndrome but only one twin pregnancy discordant fortrisomy 13 and none with trisomy 18. For this reason, it isnot possible to make definitive assessments of clinicalsensitivity for those trisomies. In addition, the trisomy 13twin pregnancy in this study had the lowest fetal fraction

Figure 1 Chromosomes 21, 18, and 13 testing results in singletonand twin pregnancies. The proportion of circulating cell-free DNAderived from the fetus/placenta (fetal fraction) is shown on thehorizontal axis. The massively parallel shotgun sequencing testingresults are reported as flow-cell adjusted chromosome z-scores(vertical axis) for chromosomes (A) 21, (B) 18, and (C) 13. Allthree sets of results were adjusted for the guanine�cytosine(GC) content bias. The chromosome 21 results were filtered withrespect to a repeat-masked genome and then adjusted for GCbias (GCRM). The dashed line at a z-score of 3 indicates theoriginal test cut-off for indicating the demarcation betweeneuploid and Down syndrome (or other aneuploid) fetuses. The solidline at a z-score of 0 indicates the average value in singleton euploidpregnancies. Five twin pregnancies discordant for Down syndromeare shown as open circles, whereas the two concordant affectedtwins are filled circles. The filled triangle indicates the one twinpregnancy discordant for trisomy 13. The 17 open diamonds areresults from euploid twin pregnancies. The open triangles indicateresults for the two euploid triplet pregnancies

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of any twin pregnancy tested. This finding may beconsistent with smaller fetal and placental size reported intrisomy 13 at mid-pregnancy (16weeks, 3 days in this case).Although in our recent study of singleton pregnancies, themedian fetal fraction for 12 cases of trisomy 13 was 13.5%,almost the same as the median for euploid singletonpregnancies (13.4%). Smaller placentas have been noted intrisomies 18 and 13 but not in trisomy 21.11–13 Further studiesof twin pregnancies with trisomies 18 and 13 are needed to reachany firm conclusions on test sensitivity for these pregnancies.Because there were no instances of trisomy among tripletpregnancies, no conclusions can be reached. On the basis ofthe two observations, however, euploid twin and tripletpregnancies may be associated with the same high specificityfound for singleton pregnancies.

Based on the results of this study, the biology of twinpregnancies, and the rationale behind MPSS testing, it isreasonable to expect that at least Down syndrome can beidentified in twin pregnancies. Test performance would besimilar to that found for singleton pregnancies, with the caveatthat the interpretation of fetal fraction at lower levels maydiffer in twin versus singleton pregnancies. This findingunderscores the usefulness of routinely quantifying fetalfraction as part of the testing and interpretive process. As weand others have suggested for the clinical implementation ofMPSS testing in women with singleton pregnancies, womenwith twin pregnancies identified as being at high risk for Down

syndrome can be offered MPSS testing to refine their risk aspart of the decision-making process. Performance ofchromosomes 18 and 13 testing in twin pregnancies shouldbe reliable but awaits further study.

ACKNOWLEDGEMENTSWe thank the Oversight Committee members and enrollmentsite personnel as well as other study personnel at SequenomCenter for Molecular Medicine, and at Women and InfantsHospital for their assistance in study activities.

WHAT’S ALREADY KNOWN ABOUT THIS TOPIC?

• Several studies, including two authored by this group and based onthe cohort used in this report, have demonstrated that applying next-generation sequencing techniques to circulating cell-free DNA frommaternal plasma can detect nearly all singleton pregnancies withDown syndrome, trisomies 18 and 13 in singleton pregnancieswith few false positive results or failures.

WHAT DOES THIS STUDY ADD?

• One previous study reported on five twin pregnancies, two withDown syndrome; this report is the largest to date to examine testperformance among twenty-five twin pregnancies, seven with Downsyndrome and one with trisomy 13.

REFERENCES1. Chiu RW, Akolekar R, Zheng YW, Leung TY, Sun H, Chan KC, Lun FM,

Go AT, Lau ET, To WW, Leung WC, Tang RY, Au-Yeung SK, Lam H, KungYY, Zhang X, van Vugt JM, Minekawa R, Tang MH, Wang J, Oudejans CB,Lau TK, Nicolaides KH, Lo YM. Non-invasive prenatal assessment oftrisomy 21 by multiplexed maternal plasma DNA sequencing: largescale validity study. BMJ 2011;342:c7401.

2. Chiu RW, Chan KC, Gao Y, Zheng W, Leung TY, Foo CH, Xie B, Tsui NB,Lun FM, Zee BC, Lau TK, Cantor CR, Lo YM. Noninvasive prenataldiagnosis of fetal chromosomal aneuploidy by massively parallel genomicsequencing of DNA in maternal plasma. Proc Natl Acad Sci USA2008;105:20458–63.

3. Ehrich M, Deciu C, Zwiefelhofer T, Tynan JA, Cagasan L, Tim R, Lu V,McCullough R, McCarthy E, Nygren AO, Dean J, Tang L, Hutchison D,Lu T, Wang H, Angkachatchai V, Oeth P, Cantor CR, Bombard A, vanden Boom D. Noninvasive detection of fetal trisomy 21 by sequencing ofDNA in maternal blood: a study in a clinical setting. Am J ObstetGynecol 2011;204:205 e201-11.

4. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasivediagnosis of fetal aneuploidy by shotgun sequencing DNA frommaternal blood. Proc Natl Acad Sci USA 2008;105:16266–71.

5. Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE, NeveuxLM, Ehrich M, van den Boom D, Bombard AT, Deciu C, Grody WW,Nelson SF, Canick JA. DNA sequencing of maternal plasma to detectDown syndrome: an international clinical validation study. Genet Med2011;13:913–20.

6. Lun FM, Jin YY, Sun H, Lau TK, Chiu RW, Lo YM. Noninvasive prenataldiagnosis of a case of Down syndrome due to robertsonian

translocation by massively parallel sequencing of maternal plasmaDNA. Clin Chem 2011;57:917–9.

7. Palomaki GE, Deciu C, Kloza EM, Lambert-Messerlian GM, HaddowJE, Neveux LM, Ehrich M, van den Boom D, Bombard AT, GrodyWW, Nelson SF, Canick JA. DNA sequencing of maternal plasmareliably identifies trisomy 18 and trisomy 13, as well as Downsyndrome: an international collaborative study. Genet Med2012;14:296–305.

8. Sehnert AJ, Rhees B, Comstock D, de Feo E, Heilek G, Burke J, Rava RP.Optimal detection of fetal chromosomal abnormalities by massivelyparallel DNA sequencing of cell-free fetal DNA from maternal blood.Clin Chem 2011;57:1042–9.

9. Pinar H, Sung CJ, Oyer CE, Singer DB. Reference values for singletonand twin placental weights. Pediatr Pathol Lab Med 1996;16:901–7.

10. Attilakos G, Maddocks DG, Davies T, Hunt LP, Avent ND, Soothill PW,Grant SR. Quantification of free fetal DNA in multiple pregnancies andrelationship with chorionicity. Prenat Diagn 2011;31:967–72.

11. Arizawa M, Nakayama M. Pathological analysis of the placenta intrisomies 21, 18 and 13. Nihon Sanka Fujinka Gakkai Zasshi1992;44:9–13.

12. Moran CJ, Tay JB, Morrison JJ. Ultrasound detection and perinataloutcome of fetal trisomies 21, 18 and 13 in the absence of a routine fetalanomaly scan or biochemical screening. Ultrasound Obstet Gynecol2002;20:482–5.

13. Shepard TH, FitzSimmons JM, Fantel AG, Pascoe-Mason J. Placentalweights of normal and aneuploid early human fetuses. Pediatr Pathol1989;9:425–31.

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